If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
* This work was supported by the Singapore Agency for Science, Technology and Research (A*STAR) Biomedical Research Council Grants 03/1/22/18/219 and 04/1/22/19/358. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The leukocyte β2 integrins are heterodimeric adhesion receptors required for a functional immune system. Many leukocyte adhesion deficiency-1 (LAD-1) mutations disrupt the expression and function of β2 integrins. Herein, we further characterized the LAD-1 mutation N329S in the β2 inserted (I)-like domain. This mutation converted αLβ2 from a resting into a high affinity conformer because αLβ2N329S transfectants adhered avidly to ligand intercellular adhesion molecule (ICAM)-3 in the absence of additional activating agent. An extended open conformation is adopted by αLβ2N329S because of its reactivity with the β2 activation reporter monoclonal antibodies MEM148 and KIM127. A corresponding mutation inβ3 generated constitutively activeαIIbβ3 that adhered to fibrinogen. This Asn is conserved in all human β subunits, and it resides before the last helix of the I-like domain, which is known to be important in activation signal propagation. By mutagenesis studies and review of existing integrin structures, we conjectured that this conserved Asn may have a primary role in shaping the I-like domain by stabilizing the conformation of theα7 helix and the β6-α7 loop in the I-like domain.
The integrins are type I membrane cell adhesion molecules formed non-covalently by two subunits (α/β). Despite having no intrinsic enzymatic properties, integrins are bidirectional signal transducers brought about by the recruitment of cytosolic proteins, many of which are signaling proteins, to their relatively short cytoplasmic tails (
The abbreviations used are: MIDAS, metal ion-dependent adhesion site; ADMIDAS, adjacent to MIDAS; LIMBS, ligand-induced metal-binding site; ICAM, intercellular adhesion molecule; LAD-1, leukocyte adhesion deficiency-1; mAb, monoclonal antibody; PBS, phosphate-buffered saline; I, inserted; EI, expression index; PDB, Protein Data Bank.
3The abbreviations used are: MIDAS, metal ion-dependent adhesion site; ADMIDAS, adjacent to MIDAS; LIMBS, ligand-induced metal-binding site; ICAM, intercellular adhesion molecule; LAD-1, leukocyte adhesion deficiency-1; mAb, monoclonal antibody; PBS, phosphate-buffered saline; I, inserted; EI, expression index; PDB, Protein Data Bank.
that contains a divalent cation essential for ligand binding. Integrins that lack the I domain (henceforth referred to as I-less integrins) are found to bind ligand via the β propeller of their α subunit and the I-like domain of their β subunit (
). The I-like domain is found in all integrin β subunits, and it is structurally similar to that of the I domain. However, it contains a specificity-determining loop, which was reported to contribute toward ligand binding specificity and integrin αβ subunit association (
), and it has two additional divalent cation-binding sites. The MIDAS of the I-like domain is flanked by the adjacent to MIDAS (ADMIDAS) and the ligand-induced metal-binding site (LIMBS), which serve as negative and positive regulatory sites, respectively (
). These integrins maintain a functional immune system by their direct involvement in processes such as leukocyte adhesion and migration, phagocytosis, and antigen presentation. All four members contain the I domain that serves as the primary ligand-binding site. The structure of an I domain-containing integrin is lacking. Thus, the mechanism of I domain regulation by other domain(s) in an intact integrin is drawn largely from isolated I domain structures without or with ligands (
). Notably, I domain ligand binding was shown to be regulated allosterically by the I-like domain. An invariant glutamate residing in the last helix of the I domain serves as an intrinsic ligand for the I-like domain (
). Ser116 and Asp209 are coordinating residues of the MIDAS/ADMIDAS and LIMBS, respectively (Fig. 1A). Interestingly, the I-like domain mutation N329S, which was not inherited from either parent, was identified in a patient having another mutation causing aberrant splicing of the integrin β2 subunit (
). Herein, we further characterize and report N329S as a mutation that promotes a high affinity αLβ2. Similarly, the same mutation introduced into the I-less integrin αIIbβ3 produced an active receptor. Combinatorial analyses of N329S with S116P or D209H showed that the activating effect of N329S requires functional I-like domain MIDAS and LIMBS to allow propagation of the activating signal to the αL I domain. Of note, this Asn at position 329 in integrin β2 primary sequence is conserved in all integrin β subunits. It may interact with neighboring residues to stabilize the β I-like domain β6-α7 loop that is required for the transmission of activation signal.
Reagents and mAbs—Recombinant human ICAM-1/Fc and ICAM-3/Fc were prepared as described previously (
). The αIIb and β3 pcDNA3 expression plasmids were kindly provided by P. J. Newman, Blood Center of Wisconsin and Medical College Wisconsin. Amino acid numbering of the integrins is based on Barclay et al. (
). All integrin mutants were generated using the QuikChange™ site-directed mutagenesis kit (Stratagene, La Jolla, CA) with the relevant primer pair. Integrins with more than one mutation (e.g. β2N329S/D209H) were generated by sequential site-directed mutagenesis. The αLc-c construct was generated by mutating I domain Lys287 and Lys294 into Cys to allow disulfide bridge formation (
). All constructs were verified by sequencing (Research Biolabs, Singapore).
Cell Transfections and Expression Analyses—Human embryonic kidney 293T cells were obtained from ATCC (Manassas, VA), and maintained in growth medium Dulbecco's modified Eagle's medium (Sigma-Aldrich) supplemented with 10%(v/v) heat-inactivated fetal bovine serum (Sigma), 100 IU/ml penicillin, and 100 μg/ml streptomycin (Sigma-Aldrich) at 37 °C in a 5% CO2 humidified atmosphere. 293T cells were transfected with expression plasmids using the calcium phosphate method (
). Cell surface expression of β3 and β2 integrins was analyzed using the mAb 7E3 and the heterodimer-specific mAb MHM23, respectively, followed by flow cytometry analysis on a FACSCalibur using the software CellQuest (BD Biosciences). The expression level was represented by the expression index (EI) that was calculated by the percentage of cells gated positive × geo-mean fluorescence intensity. An irrelevant mAb was used as background control in all preparations.
Cell Adhesion Assays—Adhesion of αLβ2 transfectants to ICAMs was performed as reported (
). Briefly, each Polysorb microtiter well (Nunc, Rosklide, Denmark) was coated with 0.5 μg of goat anti-human IgG (Fc specific) (Sigma) in 100 μl of 50 mm bicarbonate buffer (pH 9.2). Nonspecific binding sites were blocked with 0.5% (w/v) bovine serum albumin in PBS for 30 min at 37 °C. Thereafter, 50 μl of ICAM-Fc at 1 ng/μl in PBS containing 0.1% (w/v) bovine serum albumin was added to each well and incubated for 2 h at room temperature. Wells were washed twice in RPMI wash buffer (RPMI medium containing 5% (v/v) heat-inactivated fetal bovine serum and 10 mm HEPES, pH 7.4) before assay. Cells labeled with 3.0 mm 2′,7′ bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein fluorescent dye (Molecular Probes, Eugene, OR) were incubated in wash buffer containing Mg/EGTA (5 mm MgCl2 and 1.5 mm EGTA) and/or activating mAb KIM185 (10 μg/ml) to activate αLβ2. αLβ2-mediated adhesion specificity was demonstrated using MHM24 (10 μg/ml). Fluorescence signal, which correlates with the number of cells adhering to the ligand-coated well, is measured using a FL600 fluorescent plate reader (Bio-Tek instruments, Winooski, VT). For αIIbβ3 transfectant adhesion to fibrinogen, fibrinogen at 1 μg/ml in PBS was added into each microtiter well. The subsequent steps in the binding assay were the same as that of αLβ2 aforementioned. αIIbβ3-mediated adhesion specificity was assessed using the function-blocking mAb 10E5 (10 μg/ml). 1 mm MnCl2 was used to activate αIIbβ3.
Surface Labeling and Immunoprecipitation—Surface labeling of integrins with biotin was described previously (
). Cells were washed once in PBS and incubated in sulfo-NHS-biotin (Pierce) at 0.5 mg/ml in PBS for 20 min on ice. The reaction was terminated by washing surface-labeled cells once in PBS containing 10 mm Tris-HCl (pH 8.0). Thereafter, labeled cells were incubated in warm culture medium containing appropriate mAb MHM23, KIM127, or MEM148 (2 μg each) in the absence or presence of Mg/EGTA for 30 min at 37 °C. Cells were spun down and lysed in lysis buffer (10 mm Tris-HCl (pH 8.0), 150 mm NaCl, and 1% (v/v) Nonidet P-40) containing appropriate protease inhibitors (Roche Diagnostics, Basel, Switzerland) followed by immunoprecipitation with rabbit anti-mouse IgG coupled to Protein A-Sepharose beads (Amersham Biosciences, Buckinghamshire, UK). Bound proteins were resolved on a 7.5% SDS-PAGE gel under reducing conditions. Proteins were transferred onto Immobilon P membrane (Millipore, Bedford, MA), and biotinylated protein bands were detected with streptavidin-horseradish peroxidase followed by enhanced chemiluminescence detection using the ECL Plus kit (Amersham Biosciences).
Structural Images and Modeling—LSQKAB (Collaborative Computational Project, CCP4) (
) was used for molecular least-squares superposition of the three I-like domain conformers. Figs. 1B and 7 were created using PyMOL. The solvent-accessible surface area of β3 Asn339 of the three conformers was calculated using AREAIMOL (Collaborative Computational Project Number 4) with a probe of 1.7 Å radius: 46.1 Å2 (conformer I); 16.5 Å2 (conformer II); 68.8 Å2 (conformer III). Structural models of wild-type β2 I-like domain or variants were generated using Modeler 9.1. Energy computations of wild-type β2 I-like domain model or variants having Asn329 substituted with Gln, Ala, Ser, Thr, or Asp were done with GROMOS96 implementation of Swiss-PdbViewer. The models generated were examined for potential hydrogen bond(s) formation between Gln329, Ala329, Ser329, Thr329, or Asp329 of the α7 helix with Ser324 and Glu322 of the β6 strand. In the wild-type β2 I-like domain, Asn329 δ1O and δ2NH2 hydrogen-bond with Ser324 and main chain carbonyl of Glu322, respectively. In variant N329Q, Gln329ϵ1O hydrogen-bonds with Ser324. The variants N329A, N329S, and N329T lack a hydrogen bond between Asn329 and Ser324 or Glu322. In variant N329D, Asp329 hydrogen-bonds with Ser324 but not with Glu322.
N329S Generates a High Affinity αLβ2 That Adheres to ICAM-1 and ICAM-3 Substrates Constitutively—The possibility of at least three affinity states of αLβ2 based on functional and structural studies prompted us to examine the affinity state of αLβ2N329S (
). Herein, αLβ2N329S transfectant showed a high level of constitutive adhesion to ICAM-1 even in the absence of activating agent, whereas wild-type αLβ2 required activation with Mg/EGTA (Fig. 2). This is consistent with our first report on the constitutive activity of αLβ2 N329S with respect to ICAM-1 adhesion (
). Importantly, unlike wild-type αLβ2 transfectant, αLβ2N329S transfectant also showed a high level of constitutive adhesion to ICAM-3 in the absence of activating agents Mg/EGTA and the integrin β2-specific activating mAb KIM185 (Fig. 2). The expressions (represented as EI) of wild-type αLβ2 and αLβ2N329S were comparable as assessed by flow cytometry using the β2 integrin heterodimer-specific mAb MHM23. The binding specificity was demonstrated using the αLβ2-specific function-blocking mAb MHM24. Thus, the mutation N329S generates a high affinity αLβ2 with respect to ICAMs adhesions.
The Requirement of Cβ Instead of Cγ Amide Functional Group at Position 329 of the β2 I-like Domain—The Asn at position 329 of the β2 is conserved in all integrin β subunits. Because the native structure of the β2 I-like domain has not been resolved and because of the fact that the corresponding residue in the β3 is Asn339, we made use of the resolved structure of the β3 I-like domain of αVβ3 in complex with an Arg-Gly-Asp (RGD) ligand (
) to visualize the position of this conserved Asn and the three metal-binding sites. The β3 Asn339 lies before the last helix of the I-like domain (Fig. 1B). Further, the position of LIMBS (gold), MIDAS (pink), and ADMIDAS (blue) cations and the location of Asp217 (coordinating residue for LIMBS) and Ser123 (coordinating residue for MIDAS) are illustrated.
In the liganded I domain, a significant downward displacement of its C-terminal helix was observed (
). Consistent with this observation, open conformation αL and αM I domains, which have high affinity ligand binding properties, were generated by introducing cystine that stabilized the last helix of the I domains (
). The substitution of an amide to a hydroxyl side chain (Asn → Ser) hints at the possibility of functional group contribution toward the marked difference in the activity of αLβ2 with β2 Asn329 or Ser329. Therefore, four other variants, αLβ2N329T, αLβ2N329Q, αLβ2N329A, and αLβ2N329D, were generated and tested for their constitutive capacities to adhere to the ICAMs (Fig. 3). All four transfectants constitutively adhered to ICAM-1 even in the absence of Mg/EGTA (Fig. 3A). Similarly, these transfectants adhered constitutively to ICAM-3 (Fig. 3B). The expression levels of αLβ2N329T, αLβ2N329Q, αLβ2N329A, and αLβ2N329D were determined by flow cytometry. The adhesion specificity was assessed by using the αLβ2-specific function-blocking mAb MHM24. It is apparent that the introduction of a hydroxyl group as a result of N329S mutation does not have a primary role in generating a constitutively active αLβ2 because the substitutions N329Q, N329A, and N329D in β2 promoted comparable ICAM-adhesion activity. It is tempting to speculate that the altered binding property of αLβ2N329S is attributed mainly to the loss of the side chain amide group at position 329 of the β2. However, the β2 mutation N329Q, which had a similar activating effect on αLβ2, suggests the requirement of Cβ instead of Cγ amide group at position 329 of the β2 I-like domain to maintain the functional integrity of αLβ2.
The LIMBS and MIDAS of the I-like Domain Are Required for the Activating Effect of N329S in I Domain-containing αLβ2—In I domain-containing integrins, the I-like domain allosterically regulates the ligand binding activity of the I domain (
). It is reasonable to suggest that structural changes at the locality of β2 N329S are propagated to the ligand-binding face of the I-like domain. This may trigger I-like domain binding of the invariant Glu in the last C-terminal helix of the αL I domain, thus activating I domain ligand binding. Therefore, disrupting the ligand-binding sites of the I-like domain should abrogate the activating signal of N329S. Indeed, transfectants bearing αLβ2 having composite mutations N329S and S116P or D209H failed to adhere to ICAM-1 even in the presence of activating agents (Fig. 4). The adhesion specificity mediated by αLβ2 was demonstrated in all cases by complete abrogation of adhesion in the presence of mAb MHM24. The expression levels of these αLβ2 variants were comparable. Ser116 is the third coordinating residue found in the signature motif DXSXS of the I-like domain MIDAS, and the function disrupting effect of S116P, identified in LAD-1 patient, has been reported (
). Collectively, these data suggest that the activating effect of N329S is propagated through the ligand-binding site(s) of the β2 I-like domain, which subsequently activates by allostery the αL I domain.
Introduction of N339S in β3, Which Corresponds to N329S in β2, Generates a Constitutively Active I-less Integrin αIIbβ3—The Asn at position 329 of integrin β2 is conserved in all integrin β subunits (Fig. 1A). To further demonstrate that the activating signal of Asn mutation is propagated to the ligand-binding site(s) of the I-like domain, we extended the investigation to the I-less integrin αIIbβ3 because the β3 I-like domain participates directly in extrinsic ligand binding (
). The corresponding Asn339 in β3 (Fig. 1B) was substituted with Ser to generate αIIbβ3N339S. Ser116 and Asp209 of the integrin β2 are also conserved in all integrin β subunits. The corresponding residues in integrin β3 are Ser123 and Asp217. Thus, the MIDAS variant αIIbβ3S123P and the LIMBS variant αIIbβ3D217H were constructed. In addition, the composite variants αIIbβ3N339S/S123P and αIIbβ3N339S/D217H were generated (Fig. 5). Expression levels of αIIbβ3 and variants on transfectants were analyzed by flow cytometry using the β3-specific mAb 7E3. Wild-type αIIbβ3 transfectants adhered avidly to its ligand fibrinogen only in the presence of activating manganese. By contrast, αIIbβ3N339S showed constitutive adhesion to fibrinogen even in the absence of manganese. The specificity of αIIbβ3-mediated adhesion was demonstrated using αIIb-specific function-blocking mAb 10E5. Substitutions S123P and D217H in β3 abolished αIIbβ3-mediated adhesion to fibrinogen. When S123P or D217H was introduced in combination with N339S, it attenuated the activating effect of N339S on αIIbβ3-mediated adhesion to fibrinogen. Therefore, we conjectured that the mutations N329S in αLβ2 and N339S in αIIbβ3 affect the function of the respective I-like domains.
An Extended Conformation of αLβ2N329S—The conversion from a severely bent to a highly extended conformation was shown to be an important event during integrin affinity state transition (
). We examined the conformation of αLβ2N329S using two β2 integrin-specific reporter mAbs. The mAb KIM127 recognizes an epitope that is masked in the β2 integrin-epidermal growth factor-2 in a bent αLβ2 conformer. This epitope is, however, presented when αLβ2 adopts an extended conformation (
M. Cheng, S. Y. Foo, M.-L. Shi, R.-H. Tang, L.-S. Kong, S. K. A. Law, and S.-M. Tan, unpublished observation.
This may complicate the analyses using the β2 reporter mAbs based on the method of flow cytometry because these mAbs will also stain the free β2. The method of surface labeling and immunoprecipitation allows us to examine heterodimer reactivity with the β2-specific reporter mAbs by detecting the level of αL co-precipitated with the β2. Cell surface αLβ2 and αLβ2N329S on transfectants were labeled with biotin and immunoprecipitated with MHM23, KIM127, or MEM148 with or without Mg/EGTA. The αLc-c was reported previously in which a disulfide bridge was engineered in the αL I domain to “lock” the I domain in an active open conformation (
) (see “Experimental Procedures”). We determined that αLc-cβ2 transfectants adhered constitutively to ICAM-1 but not ICAM-3, suggesting an intermediate affinity conformer (data not shown). Thus, αLc-cβ2 was included for comparison with αLβ2N329S on conformational changes.
Wild-type αLβ2 was precipitated by MHM23 (a β2 integrin heterodimer-specific mAb that serves as a control in this experiment) (Fig. 6). However, wild-type αLβ2 was only precipitated by KIM127 or MEM148 in the presence of activating Mg/EGTA. A similar profile was detected for αLc-cβ2. By contrast, αLβ2N329S was precipitated by KIM127 and MEM148 even in the absence of Mg/EGTA. These data suggest that αLβ2N329S adopts an extended conformation with possible displacement of the hybrid domain. This conformation was proposed to depict a high affinity integrin (
), and it is in agreement with our binding data. The lack of αLc-cβ2 precipitated by KIM127 in the absence of exogenous activation was also in parallel with the previous observation that αLc-cβ2 was stained weakly with KIM127 by flow cytometry (
). This study further characterized the N329S mutation. The β2 Asn329 is conserved in all human integrin β subunits (Fig. 1A) and in the β subunits of several other metazoans including coral and sponge (
). Our data suggest that a Cβ instead of Cγ amide group at position 329 of the β2 I-like domain is required to maintain αLβ2 functional integrity. Because of the availability of structural information of the β3 I-like domain, we first reviewed Asn339 in β3 that corresponds to the Asn329 in β2. The β3 Asn339 is located before the α7 helix (last helix) of its I-like domain (Fig. 7A). The orientations of the β6 strand and the α7 helix in the β3 I-like domain are different in the structures of a bent αVβ3 (red) (conformer I) (
). The structure of the bent-liganded αVβ3 may require careful interpretation because the αVβ3 crystals were soaked in the RGD ligand instead of co-crystallization of an RGD and αVβ3 protein mixture, and no displacement of the α7 helix was observed in the structure (
). Thus, the repositioning of the Asn339 herein was examined mainly between conformer I and conformer III. In conformer I, Asn339 of the β3 I-like domain projects its amide side chain in an orientation that can allow hydrogen bonds to be established with the main chain carbonyl oxygen of Val332 in the β6 strand and the side chain hydroxyl group of Ser334 that resides in the loop connecting the β6 strand and the α7 helix. Interestingly, these are different in conformer III. Hydrogen bond can form between δ1O of Asn339 with the main chain –NH of Val332 but not between Asn339 and Ser334. This observed difference between the two conformers is attributed to the downward movement of the α7 helix and the outward rotation of the Asn339 in conformer III with respect to conformer I (Fig. 7B). The solvent-accessible surface area of Asn339 of conformer I is 46.1 Å2, whereas that of conformer III is 68.8 Å2, which corroborates well with its outward reorientation in conformer III having a displaced α7 helix. It is, therefore, possible that the formation of polar contacts between Asn339 with Val332 and Ser334 is required in part to maintain β3 I-like domain in a resting conformation. Disruption of these contacts as a result of a mutation such as N339S may destabilize these interactions, thus generating an activated αIIbβ3.
In β2, similar polar contacts may be formed as depicted in a homology model of β2 I-like domain generated based on the bent αVβ3 structural coordinates (Fig. 7C). The Ser324 side chain may hydrogen-bond with Asn329 δ1Ointhe β2 I-like domain model. The residue at position 322 of β2 and 332 of β3 is different. This is, however, irrelevant because it is the backbone carbonyl of this residue that may hydrogen-bond with the conserved Asn. β2 I-like domain models incorporating Asn329 substituted with Gln, Ala, Ser, Thr, or Asp (see “Experimental Procedures”) showed potential disruptions in hydrogen-bond formation between these residues at position 329 with Ser324 and main chain Glu322, which may account for the activating effect of these mutations on αLβ2. Together, these suggest that the polar contacts between Asn329 with Ser324 and Glu322 may serve to stabilize the β2 I-like domain in a fashion similar to that in β3 conformer I. It is noted that although β2 Ser324 (β3 Ser334) can potentially hydrogen-bond with the conserved β2 Asn329(β3 Asn339), this should be extrapolated with caution to other integrins because β2 Ser324 is conserved in β3, β1, and β7 but not in other β subunits (Fig. 1A). In addition, the contribution of the conserved Asn toward the function of other β subunits awaits further studies.
Integrin affinity states are governed by its varied conformations under different conditions (
). Extended integrin conformers with or without hybrid domains displaced are assigned high or intermediate affinity states, respectively. In this study, the mutation N329S induced an extended αLβ2 conformation because in the absence of activating agent, αLβ2N329S was immunoprecipitated by the reporter mAb KIM127 (
). These data suggest a high affinity αLβ2 generated by the β2N329S mutation. Indeed, our functional data showing αLβ2N329S binding constitutively and effectively to ICAM-3 supported the assignment of a high affinity receptor. It is unclear at present how N329S triggers such a dramatic global conformational change in αLβ2. Not only does N329S incur activation of the I-like domain, as demonstrated in αIIbβ3N339S, it also induces unbending of the entire integrin. However, this need not be unexpected because it was reported that one-turn deletion in the α7 helix of the β2 I-like domain promoted αLβ2 binding to ICAM-1 and exposed the epitopes of extension reporter mAb KIM127 and the activated I-like domain reporter mAb m24 (
In summary, we have further characterized the LAD-1 mutation N329S. This mutation generates a high affinity αLβ2 with an extended conformation. N329S activates the β2 I-like domain. The corresponding mutation in β3 N339S had similar activating effect on αIIbβ3. The conservation of this Asn in all human integrin β subunits through evolution suggests a primary role of this residue in the shaping of the I-like domain. Based on available structures of the β I-like domains and structural predictions, this Asn may serve to stabilize the conformation of the α7 helix and its interaction with the preceding β6 strand. However, definitive revelation of its role in the shaping of the I-like domain will require structural studies of integrins incorporating the aforementioned mutation(s).
We thank Elianna Bte Mohamed Amin, and Manisha Cooray for technical support.